Enabling Multiple Conjugation to Oligonucleotides Using "Click Cycles".

An efficient method for the synthesis of multiply functionalized oligonucleotides (ONs) utilizing a novel H-phosphonate alkyne-based linker for multiple functionalization (LMF) is developed. The strategy allows for the conjugation of various active entities to oligonucleotide through the postsynthetic attachment of LMF at the 5'-terminus of ONs using H-phosphonate chemistry followed by conjugation of various entities via [3 + 2] copper(I) catalyzed cycloaddition in a stepwise manner. Each cycle is composed of attachment of the LMF followed by a click reaction with azido-containing units. Sequential solid-phase synthesis of oligonucleotide conjugates containing three attached entities was performed using an acetylated form of MIF peptide conjugated to azido linker, achieving high conversions at each unit addition. In addition, to show the versatility of the method, oligonucleotide conjugates with several different classes of compounds were synthesized. Each conjugate containing three different entities, whose structure and function varied (e.g., sugars, peptides, fluorescent labels, and m3G-Caps).

Synthesis of biotin linkers with the activated triple bond donor [p-(N-propynoylamino)toluic acid] (PATA) for efficient biotinylation of peptides and oligonucleotides.

Biotin is an important molecule for modern biological studies including, e.g., cellular transport. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows ready and specific detection. As a consequence methods for the attachment of biotin to various biological targets are of high importance, especially when they are very selective and can also proceed in water. One useful method is Hüisgen dipolar [3+2]-cycloaddition, commonly referred to as “click chemistry”. As we reported recently, the activated triple bond donor p-(N-propynoylamino)toluic acid (PATA) gives excellent results when used for conjugations at submicromolar concentrations. Thus, we have designed and synthesized two biotin linkers, with different lengths equipped with this activated triple bond donor and we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution and on solid support with excellent yields of conversion.

Palladium-catalyzed propargylic substitution with phosphorus nucleophiles: efficient, stereoselective synthesis of allenylphosphonates and related compounds.

A new, efficient method is developed, based on a palladium(0)-catalyzed reaction of propargylic derivatives with various phosphorus nucleophiles, to produce allenylphosphonates and their analogues with defined stereochemistry in the allenic and the phosphonate moiety.